The discovery of the single gene (CFTR) cause of cystic fibrosis (CF) has been transformational, focusing treatment on modulator drugs that restore function of the CFTR gene product. CFTR correctors enhance transport of mutant protein to the cell membrane and CFTR potentiates activate mutant protein that does get to the membrane. The FDA-approved potentiate drug invocator has resulted in remarkable quality of life improvements for the ~5% of the CF population with specific, responsive CFTR mutations. Mixed results, but none approaching the invocator outcome, have been achieved for the remaining majority of the CF population. The major hurdle is that over 2000 different CFTR gene mutations have been identified, some relatively frequent and others quite rare, so that available cell-based assays for testing potential drugs have unpredictable relevance for an individual with CF. In the absence of cell-based ex vivo or any known in vivo assays, there are no reliable predictors of clinical outcomes specific to the individual, leading to arduous, potentially harmful, trial and error drug treatment without any predictive basis for success. This STTR proposal seeks to overcome this hurdle by developing a personalized, ex vivo, minimally invasive, and predictive screening assay for CFTR modulator activity. Cells from a single, minimally invasive nasal biopsy are cultured to generate many nasospheres, three-dimensional cultures that remarkably and relatively quickly replicate the nasal epithelium with a ciliated linig and a mucus barrier. Each nasosphere constitutes an ex vivo organoid, a replica of the donor's nasal cells, amenable to drug testing, detailed probing of outcome measures and precise measurement of restored CFTR activity. The ability to generate many nasospheres from a single biopsy allows combinatorial drug testing of restored CFTR activity for that person, and the potential to optimize all available drugs for an individual. Accepted outcome measures that correlate with restored CFTR activity are nasosphere volume change and cilia beat frequency. Further, we propose novel measurements of nasosphere mucus viscoelasticity using particle tracking microbeads rheology, which reflects changes in mucus hydration and is a predictor of cilia propulsion of mucus and mucus clearance. Phase 1 success will lead to Phase 2 in which nasosphere assay outcome measures will be correlated with in vivo drug treatment efficacy. Success in Phase 2 will lead to commercialization of the nasosphere assay and associated data analysis to enable urgently required personalized, reliable, and predictive treatment decisions for individuals with CF.